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July 7, 2019  |  

GAML: genome assembly by maximum likelihood.

Resolution of repeats and scaffolding of shorter contigs are critical parts of genome assembly. Modern assemblers usually perform such steps by heuristics, often tailored to a particular technology for producing paired or long reads.We propose a new framework that allows systematic combination of diverse sequencing datasets into a single assembly. We achieve this by searching for an assembly with the maximum likelihood in a probabilistic model capturing error rate, insert lengths, and other characteristics of the sequencing technology used to produce each dataset. We have implemented a prototype genome assembler GAML that can use any combination of insert sizes with Illumina or 454 reads, as well as PacBio reads. Our experiments show that we can assemble short genomes with N50 sizes and error rates comparable to ALLPATHS-LG or Cerulean. While ALLPATHS-LG and Cerulean require each a specific combination of datasets, GAML works on any combination.We have introduced a new probabilistic approach to genome assembly and demonstrated that this approach can lead to superior results when used to combine diverse set of datasets from different sequencing technologies. Data and software is available at http://compbio.fmph.uniba.sk/gaml.


July 7, 2019  |  

Complete and assembled genome sequence of Staphylococcus aureus RKI4, a food-poisoning strain exhibiting a novel S. aureus pathogenicity island carrying seb.

The genome of Staphylococcus aureus RKI4, a strain isolated from feces of a patient in a case of staphylococcal food poisoning, was sequenced using combined Illumina and single-molecule real-time sequencing. Hierarchical assembly of the genome resulted in a 2,725,654-bp chromosome and a 17,905-bp mobile genetic element. Copyright © 2015 Stevens et al.


July 7, 2019  |  

Identification and heterologous expression of the chaxamycin biosynthetic gene cluster from Streptomyces leeuwenhoekii.

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ?cxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

First complete genome sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 (DSM 20231T), determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 strain DSM 20231(T), the type strain of the bacterium causing staphylococcal disease, were determined using PacBio RS II. The sequences represent the chromosome (2,755,072 bp long; G+C content, 32.86%) and a plasmid (27,490 bp long; G+C content, 30.69%). Copyright © 2015 Shiroma et al.


July 7, 2019  |  

Whole-genome mapping as a novel high-resolution typing tool for Legionella pneumophila.

Legionella is the causative agent for Legionnaires’ disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection.Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n?=?174) and without rifampicin (n?=?126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains.Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).


July 7, 2019  |  

Insights on virulence from the complete genome of Staphylococcus capitis.

Staphylococcus capitis is an opportunistic pathogen of the coagulase negative staphylococci (CoNS). Functional genomic studies of S. capitis have thus far been limited by a lack of available complete genome sequences. Here, we determined the closed S. capitis genome and methylome using Single Molecule Real Time (SMRT) sequencing. The strain, AYP1020, harbors a single circular chromosome of 2.44 Mb encoding 2304 predicted proteins, which is the smallest of all complete staphylococcal genomes sequenced to date. AYP1020 harbors two large mobile genetic elements; a plasmid designated pAYP1020 (59.6 Kb) and a prophage, FAYP1020 (48.5 Kb). Methylome analysis identified significant adenine methylation across the genome involving two distinct methylation motifs (1972 putative 6-methyladenine (m6A) residues identified). Putative adenine methyltransferases were also identified. Comparative analysis of AYP1020 and the closely related CoNS, S. epidermidis RP62a, revealed a host of virulence factors that likely contribute to S. capitis pathogenicity, most notably genes important for biofilm formation and a suite of phenol soluble modulins (PSMs); the expression/production of these factors were corroborated by functional assays. The complete S. capitis genome will aid future studies on the evolution and pathogenesis of the coagulase negative staphylococci.


July 7, 2019  |  

Methicillin-susceptible, vancomycin-resistant Staphylococcus aureus, Brazil.

We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.


July 7, 2019  |  

Role of restriction-modification systems in prokaryotic evolution and ecology

Restriction–modification (R-M) systems are able to methylate or cleave DNA depending on methylation status of their recognition site. It allows them to protect bacterial cells from invasion by foreign DNA. Comparative analysis of a large number of available bacterial genomes and methylomes clearly demonstrates that the role of R-M systems in bacteria is wider than only defense. R-M systems maintain heterogeneity of a bacterial population and are involved in adaptation of bacteria to change in their environmental conditions. R-M systems can be essential for host colonization by pathogenic bacteria. Phase variation and intragenomic recombinations are sources of the fast evolution of the specificity of R-M systems. This review focuses on the influence of R-M systems on evolution and ecology of prokaryotes.


July 7, 2019  |  

A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain.

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form. Copyright © 2015 Elsevier B.V. All rights reserved.


July 7, 2019  |  

Enzymatic degradation of phenazines can generate energy and protect sensitive organisms from toxicity.

Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed.Phenazine production by Pseudomonas spp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnover in situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines. Copyright © 2015 Costa et al.


July 7, 2019  |  

Twenty years of bacterial genome sequencing.

Twenty years ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of bacterial genome sequencing, which have been marked by three revolutions: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a ‘sequencing singularity’, where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.


July 7, 2019  |  

Genome analysis of Staphylococcus agnetis, an agent of lameness in broiler chickens.

Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.


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